Diagnostic test system using measurement obtained from reference feature to modify operational parameter of reader

ABSTRACT

A diagnostic test system includes a housing, a reader, and a data analyzer. The housing includes a port constructed and arranged to receive a test strip that includes a flow path for a fluid sample, a sample receiving zone couple to the flow path, a label that specifically binds a target analyte, a detection zone coupled to the flow path and comprising a test region exposed for optical inspection and having an immobilized test reagent that specifically binds the target analyte, and at least one reference feature. The reader is operable to obtain light intensity measurements from exposed regions of the test strip when the test strip is loaded in the port. The data analyzer is operable to perform operations including at least one of (a) identifying ones of the light intensity measurements obtained from the test region based on at least one measurement obtained from the at least one reference feature, and (b) generating a control signal modifying at least one operational parameter of the reader based on at least one measurement obtained from the at least one reference feature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/296,872, filed Oct. 18, 2016 and scheduled to issue as U.S. Pat. No.10,191,043 on Jan. 29, 2019, which is a continuation of U.S. applicationSer. No. 12/409,877, filed Mar. 24, 2009, which is a divisional of U.S.application Ser. No. 11/280,640, filed Nov. 16, 2005 and issued as U.S.Pat. No. 7,521,260 on Apr. 21, 2009, which is a continuation-in-part ofU.S. application Ser. No. 11/112,807, filed Apr. 22, 2005 and issued asU.S. Pat. No. 8,128,871 on Mar. 6, 2012, all of which are incorporatedby reference in their entirety.

BACKGROUND

Assay test kits are currently available for testing for a wide varietyof medical and environmental conditions or compounds, such as a hormone,a metabolite, a toxin, or a pathogen-derived antigen. FIG. 1 shows atypical lateral flow test strip 10 that includes a sample receiving zone12, a labeling zone 14, a detection zone 15, and an absorbent zone 20 ona common substrate 22. These zones 12-20 typically are made of amaterial (e.g., chemically-treated nitrocellulose) that allows fluid toflow from the sample receiving zone 12 to the absorbent zone 22 bycapillary action. The detection zone 15 includes a test region 16 fordetecting the presence of a target analyte in a fluid sample and acontrol region 18 for indicating the completion of an assay test.

FIGS. 2A and 2B show an assay performed by an exemplary implementationof the test strip 10. A fluid sample 24 (e.g., blood, urine, or saliva)is applied to the sample receiving zone 12. In the example shown inFIGS. 2A and 2B, the fluid sample 24 includes a target analyte 26 (i.e.,a molecule or compound that can be assayed by the test strip 10).Capillary action draws the liquid sample 24 downstream into the labelingzone 14, which contains a substance 28 for indirect labeling of thetarget analyte 26. In the illustrated example, the labeling substance 28consists of an immunoglobulin 30 with a detectable particle 32 (e.g., areflective colloidal gold or silver particle). The immunoglobulin 30specifically binds the target analyte 26 to form a labeled targetanalyte complex. In some other implementations, the labeling substance28 is a non-immunoglobulin labeled compound that specifically binds thetarget analyte 26 to form a labeled target analyte complex.

The labeled target analyte complexes, along with excess quantities ofthe labeling substance, are carried along the lateral flow path into thetest region 16, which contains immobilized compounds 34 that are capableof specifically binding the target analyte 26. In the illustratedexample, the immobilized compounds 34 are immunoglobulins thatspecifically bind the labeled target analyte complexes and therebyretain the labeled target analyte complexes in the test region 16. Thepresence of the labeled analyte in the sample typically is evidenced bya visually detectable coloring of the test region 16 that appears as aresult of the accumulation of the labeling substance in the test region16.

The control region 18 typically is designed to indicate that an assayhas been performed to completion. Compounds 35 in the control region 18bind and retain the labeling substance 28. The labeling substance 28typically becomes visible in the control region 18 after a sufficientquantity of the labeling substance 28 has accumulated. When the targetanalyte 26 is not present in the sample, the test region 16 will not becolored, whereas the control region 18 will be colored to indicate thatassay has been performed. The absorbent zone 20 captures excessquantities of the fluid sample 24.

In the non-competitive-type of lateral flow assay test strip designsshown in FIGS. 2A and 2B, an increase in the concentration of theanalyte in the sample results in an increase in the concentration oflabels in the test region. Conversely, in competitive-type of lateralflow assay test strip designs, an increase in the concentration of theanalyte in the fluid sample results in a decrease in the concentrationof labels in the test region.

Although visual inspection of lateral flow assay devices of the typedescribed above are able to provide qualitative assay results, such amethod of reading these types of devices is unable to providequantitative assay measurements and therefore is prone to interpretationerrors. Automated and semi-automated lateral flow assay readers havebeen developed in an effort to overcome this deficiency.

In one approach, a portable lateral flow assay reader performs assays onbodily fluids to detect the presence of certain hormones, glucose, orother bodily fluids of interest. Membrane test strips containing a fluidsample are inserted directly into a receiving port of a reader. Thereceiving port is shielded to improve sensitivity and reduce the entryof stray or ambient light into the reader. The reader includes a lightsource and one or more sensors that detect the intensity of lightreflected from the detection zones of the test strips that are insertedinto the receiving port.

In another approach, a reader detects an intensity of a detection signalarising in one or more measurement zones in a detection zone of alateral flow assay test strip as a result of the presence of animmobilized labeled target analyte complex. The reader generates abaseline of signal intensity from the measurement zones by interpolatingbetween values of the detection signal outside of the measurement zonesand inside of the detection zone. The reader quantifies a value ofsignal intensity representative of the immobilized labeled targetanalyte complex with respect to the baseline. In this process, thereader locates a beginning boundary and an ending boundary for the oneor more measurement zones on the test strip, allowing an automatic orsemi-automatic analytical instrument, or a human reader, to determinecertain results of the lateral flow assay. The signals from themeasurement zones are quantified or compared with respect to thebaseline. Quantified values corresponding to the respectiveconcentration of compounds in different measurement zones may then becompared with one another to detect the presence of antigens in thesample.

The measurements that are made by the above-described lateral flow assayreaders are based on signals from regions of the test strips thattypically are significantly larger than the regions of interest. As aresult, these measurements tend to have high noise levels and,consequently, these measurements may yield inaccurate or incorrectresults when low concentrations of analytes are involved.

SUMMARY

In one aspect, the invention features an assay test strip that includesa flow path for a fluid sample, a sample receiving zone, a label, adetection zone, and at least one position marker. The sample receivingzone is coupled to the flow path. The label specifically binds a targetanalyte. The detection zone is coupled to the flow path and includes aregion of interest and an immobilized test reagent that specificallybinds the target analyte. The at least one position marker is alignedwith respect to the region of interest such that location of the atleast one position marker indicates a position of the region ofinterest.

In one aspect, the invention features an assay test strip that includesa flow path for a fluid sample, a sample receiving zone, a label, adetection zone, and at least one reference feature. The sample receivingzone is coupled to the flow path. The label specifically binds a targetanalyte. The detection zone is coupled to the flow path and includes animmobilized test reagent that specifically binds the target analyte. Theat least one reference feature is exposed for optical inspection and hasa calibrated amount of the label.

In another aspect, the invention features a diagnostic test system thatincludes a housing, a reader, and a data analyzer. The housing includesa port constructed and arranged to receive a test strip. The test stripincludes a flow path for a fluid sample, a sample receiving zone coupledto the flow path, a label that specifically binds a target analyte, adetection zone, and at least one reference feature. The detection zoneis coupled to the flow path and includes a test region. The test regionis exposed for optical inspection and has an immobilized test reagentthat specifically binds the target analyte. The reader is operable toobtain light intensity measurements from exposed regions of the teststrip when the test strip is loaded in the port. The data analyzer isoperable to perform operations including at least one of (a) identifyingones of the light intensity measurements obtained from the test regionbased on at least one measurement obtained from the at least onereference feature, and (b) generating a control signal modifying atleast one operational parameter of the reader based on at least onemeasurement obtained from the at least one reference feature.

The invention also features a diagnostic test method in accordance withwhich a test strip is received. The test strip includes a flow path fora fluid sample, a sample receiving zone coupled to the flow path, alabel that specifically binds a target analyte, a detection zone, and atleast one reference feature. The detection zone is coupled to the flowpath and includes a test region. The test region is exposed for opticalinspection and has an immobilized test reagent that specifically bindsthe target analyte. Light intensity measurements are obtained fromexposed regions of the test strip. At least one of the following isperformed: (a) identifying ones of the light intensity measurementsobtained from the test region based on at least one measurement obtainedfrom the at least one reference feature, and (b) generating a controlsignal modifying the obtaining of light intensity measurements fromexposed regions of the test strip based on at least one measurementobtained from the at least one reference feature.

Other features and advantages of the invention will become apparent fromthe following description, including the drawings and the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagrammatic view of a prior art implementation of an assaytest strip.

FIG. 2A is a diagrammatic view of a fluid sample being applied to anapplication zone of the assay test strip shown in FIG. 1 .

FIG. 2B is a diagrammatic view of the assay test strip shown in FIG. 2Aafter the fluid sample has flowed across the test strip to an absorptionzone.

FIG. 3 is a block diagram of an embodiment of a test strip that isloaded into an embodiment of a diagnostic test system.

FIG. 4 is a flow diagram of an embodiment of a diagnostic test method.

FIG. 5A is a diagrammatic side view of an implementation of thediagnostic test system shown in FIG. 3 that includes a two-dimensionallight detector array obtaining light intensity measurements from regionsof a test strip.

FIG. 5B is a diagrammatic view of the two-dimensional light detectorarray shown in FIG. 5A in which ones of the light detectors that arepositioned to obtain light intensity measurements from the test regionand the control region are highlighted.

FIG. 6 is an exemplary graph of light intensity plotted as a function ofposition in the two-dimensional light detector array shown in FIGS. 5Aand 5B.

FIG. 7 is a flow diagram of an embodiment of a method of reading a teststrip having at least one position marker.

FIG. 8 is a flow diagram of an embodiment of a method of identifyinglight intensity measurements that are obtained from a region of interestbased on light intensity measurements that are obtained from at leastone optical position marker.

FIG. 9 is a diagrammatic view of an implementation of the test stripshown in FIG. 3 .

FIG. 10 is a diagrammatic view of an implementation of the test stripshown in FIG. 3 .

FIG. 11 is a diagrammatic view of an implementation of the test stripshown in FIG. 3 .

FIG. 12A is a diagrammatic view of an implementation of the test stripshown in FIG. 3 .

FIG. 12B is a graph of light intensity plotted as a function of positionalong the test strip shown in FIG. 12A.

FIG. 13A is a diagrammatic view of an implementation of the test stripshown in FIG. 3 .

FIG. 13B is a diagrammatic view of a detection system on a portion ofthe test strip shown in FIG. 13A.

FIG. 13C is a diagrammatic view of the detection system on a differentportion of the test strip shown in FIG. 13B.

FIG. 14A is a diagrammatic view of an implementation of the test stripshown in FIG. 3 .

FIG. 14B is a diagrammatic view of a detection system on a portion ofthe test strip shown in FIG. 14A.

FIG. 14C is a diagrammatic view of the detection system on a differentportion of the test strip shown in FIG. 14B.

FIG. 15 is a flow diagram of an embodiment of a method of calibratinglight intensity measurements that are obtained from a region ofinterest.

FIG. 16 is a diagrammatic view of an implementation of the diagnostictest system shown in FIG. 3 .

FIG. 17 is a flow diagram of an embodiment of a method of calibrating anillumination source of a diagnostic test system.

DETAILED DESCRIPTION

In the following description, like reference numbers are used toidentify like elements. Furthermore, the drawings are intended toillustrate major features of exemplary embodiments in a diagrammaticmanner. The drawings are not intended to depict every feature of actualembodiments nor relative dimensions of the depicted elements, and arenot drawn to scale.

I. INTRODUCTION

The embodiments that are described in detail below provide lateral flowassay test strips that have one or more reference features. Theseembodiments also provide diagnostic test systems that are configured toread such test strips in ways that improve the accuracy and precisionwith which analytes in a fluid sample may be assayed.

In some embodiments, the reference features are position markers thatare aligned with respect to regions of interest in the test strip. Theseembodiments enable the levels of noise (e.g., noise caused by reflectionof light or intrinsic fluorescence from materials in the test strip) inassay measurements to be reduced by restricting the measurements to theregions of interest on the test strip based on measurements obtainedfrom the reference features. In this way, these embodiments increase thesignal-to-noise levels of these measurements and, thereby, increasemeasurement sensitivity and reduce the incidence of erroneous resultsfor low concentrations of analytes.

In some embodiments, the reference features are calibration regions thatprovide a reference optical response that may be used by embodiments ofthe diagnostic test system to calibrate one or more components of adiagnostic test system and the assay measurements obtained by such asystem and, thereby, increase the accuracy of the lateral flow assayresults.

The terms “assay test strip” and “lateral flow assay test strip”encompass both competitive and non-competitive types of lateral flowassay test strips. A lateral flow assay test strip generally includes asample receiving zone and a detection zone, and may or may not have alabeling zone. In some implementations, a lateral flow assay test stripincludes a sample receiving zone that is located vertically above alabeling zone, and additionally includes a detection zone that islocated laterally downstream of the labeling zone.

The term “analyte” refers to a substance that can be assayed by the teststrip. Examples of different types of analytes include organic compounds(e.g., proteins and amino acids), hormones, metabolites, antibodies,pathogen-derived antigens, drugs, toxins, and microorganisms (e.g.,bacteria and viruses).

As used herein the term “label” refers to a substance that has specificbinding affinity for an analyte and that has a detectable characteristicfeature that can be distinguished from other elements of the test strip.The label may include a combination of a labeling substance (e.g., afluorescent particle, such as a quantum dot) that provides thedetectable characteristic feature and a probe substance (e.g., animmunoglobulin) that provides the specific binding affinity for theanalyte. In some implementations, the labels have distinctive opticalproperties, such as luminescence (e.g., fluorescence) or reflectiveproperties, which allow regions of the test strip containing differentlabels to be distinguished from one another.

The term “reagent” refers to a substance that reacts chemically orbiologically with a target substance, such as a label or an analyte.

The term “capture region” refers to a region on a test strip thatincludes one or more immobilized reagents.

The term “test region” refers to a capture region containing animmobilized reagent with a specific binding affinity for an analyte.

The term “control region” refers to a capture region containing animmobilized reagent with a specific binding affinity for a label.

II. DIAGNOSTIC TEST SYSTEM ARCHITECTURE

A. Overview

FIG. 3 shows an embodiment of a diagnostic test system 40 that includesa housing 42, a reader 44, a data analyzer 46, and a memory 47. Thehousing 42 includes a port 48 for receiving a test strip 50. When thetest strip 50 is loaded in the port 48, the reader 44 obtains lightintensity measurements from the test strip 50. In general, the lightintensity measurements may be unfiltered or they may be filtered interms of at least one of wavelength and polarization. The data analyzer46 computes at least one parameter from one or more of the lightintensity measurements. A results indicator 52 provides an indication ofone or more of the results of an assay of the test strip 50. In someimplementations, the diagnostic test system 40 is fabricated fromrelatively inexpensive components enabling it to be used for disposableor single-use applications.

The housing 42 may be made of any one of a wide variety of materials,including plastic and metal. The housing 42 forms a protective enclosurefor the reader 44, the data analyzer 46, the power supply 54, and othercomponents of the diagnostic test system 40. The housing 42 also definesa receptacle that mechanically registers the test strip 50 with respectto the reader 44. The receptacle may be designed to receive any one of awide variety of different types of test strips 50, including test stripsof the type shown in FIG. 1 .

In the illustrated embodiments, each of the test strips 50 is anon-competitive type of assay test strip that supports lateral flow of afluid sample along a lateral flow direction 51 and includes a labelingzone containing a labeling substance that binds a label to a targetanalyte and a detection zone that includes at least one test regioncontaining an immobilized substance that binds the target analyte. Oneor more areas of the detection zone, including at least a portion of thetest region, are exposed for optical inspection by the reader 44. Theexposed areas of the detection zone may or may not be covered by anoptically transparent window.

In other embodiments, the test strips are competitive type of lateralflow assay test strips in which the concentrations of the label in thetest region decreases with increasing concentration of the targetanalyte in the fluid sample. Some of these embodiments include alabeling zone, whereas others of these implementations do not include alabeling zone.

Some of these competitive lateral flow assay test strip embodimentsinclude a labeling zone that contains a label that specifically bindstarget analytes in the fluid sample, and a test region that containsimmobilized target analytes as opposed to immobilized test reagents(e.g., antibodies) that specifically bind any non-bound labels in thefluid sample. In operation, the test region will be labeled when thereis no analyte present in the fluid sample. However, if target analytesare present in the fluid sample, the fluid sample analytes saturate thelabel's binding sites in the labeling zone, well before the label flowsto the test region. Consequently, when the label flows through the testregion, there are no binding sites remaining on the label, so the labelpasses by and the test region remains unlabeled.

In other competitive lateral flow assay test strip embodiments, thelabeling zone contains only pre-labeled analytes (e.g., gold adhered toanalyte) and the test region contains immobilized test reagents with anaffinity for the analyte. In these embodiments, if the fluid samplecontains unlabeled analyte in a concentration that is large compared tothe concentration of the pre-labeled analyte in the labeling zone, thenlabel concentration in the test region will appear proportionatelyreduced.

The reader 44 includes one or more optoelectronic components foroptically inspecting the exposed areas of the detection zone of the teststrip 50. In some implementations, the reader 44 includes at least onelight source and at least one light detector. In some implementations,the light source may include a semiconductor light-emitting diode andthe light detector may include a semiconductor photodiode. Depending onthe nature of the label that is used by the test strip 50, the lightsource may be designed to emit light within a particular wavelengthrange or light with a particular polarization. For example, if the labelis a fluorescent label, such as a quantum dot, the light source may bedesigned to illuminate the exposed areas of the detection zone of thetest strip 50 with light in a wavelength range that induces fluorescentemission from the label. Similarly, the light detector may be designedto selectively capture light from the exposed areas of the detectionzone. For example, if the label is a fluorescent label, the lightdetector may be designed to selectively capture light within thewavelength range of the fluorescent light emitted by the label or withlight of a particular polarization. On the other hand, if the label is areflective-type label, the light detector may be designed to selectivelycapture light within the wavelength range of the light emitted by thelight source. To these ends, the light detector may include one or moreoptical filters that define the wavelength ranges or polarizations axesof the captured light.

The data analyzer 46 processes the light intensity measurements that areobtained by the reader 44. In general, the data analyzer 46 may beimplemented in any computing or processing environment, including indigital electronic circuitry or in computer hardware, firmware, orsoftware. In some embodiments, the data analyzer 46 includes a processor(e.g., a microcontroller, a microprocessor, or ASIC) and ananalog-to-digital converter. In the illustrated embodiment, the dataanalyzer 46 is incorporated within the housing 42 of the diagnostic testsystem 40. In other embodiments, the data analyzer 46 is located in aseparate device, such as a computer, that may communicate with thediagnostic test system 40 over a wired or wireless connection.

In general, the results indicator 52 may include any one of a widevariety of different mechanisms for indicating one or more results of anassay test. In some implementations, the results indicator 52 includesone or more lights (e.g., light-emitting diodes) that are activated toindicate, for example, a positive test result and the completion of theassay test (i.e., when sufficient quantity of labeling substance 28 hasaccumulated in the control region). In other implementations, theresults indicator 52 includes an alphanumeric display (e.g., a two orthree character light-emitting diode array) for presenting assay testresults.

A power supply 54 supplies power to the active components of thediagnostic test system 40, including the reader 44, the data analyzer46, and the results indicator 52. The power supply 54 may be implementedby, for example, a replaceable battery or a rechargeable battery. Inother embodiments, the diagnostic test system may be powered by anexternal host device (e.g., a computer connected by a USB cable).

FIG. 4 shows an embodiment of a diagnostic test method that isexecutable by the implementations of the diagnostic test system 40described below. In accordance with this method, the reader 44 obtainsseparable localized light intensity measurements from regions of theexposed area of the detection zone of the test strip 50 when the teststrip 50 is loaded in the port 48 of the diagnostic test system 40 (FIG.4 , block 60). As used herein, the term “separable localized lightintensity measurements” refers to the ability of the reader 44 totransmit or record the light intensity measurements from respectivelocalized regions of the test strip in a way that allows the dataanalyzer 46 to individually analyze each of the light intensitymeasurements.

In some embodiments in accordance with the invention, each of theseparable localized regions from which the light intensity measurementsare obtained by the reader 44 is characterized by at least one surfacedimension that is smaller than the dimension of the exposed area of thedetection zone that is transverse to the lateral flow direction 51. Insome implementations, each of these localized regions has a surfacedimension that is approximately the same size or smaller than thenarrowest dimension of a region of interest in the detection zone 15(e.g., the test region, the control region, or a region of animmobilized labeled or unlabeled complex).

After the reader 44 has obtained light intensity measurements from suchlocalized regions of interest in the detection zone 15 (FIG. 4 , block60), the data analyzer 46 identifies ones of the light intensitymeasurements obtained from the regions of interest (FIG. 4 , block 62).In this process, the data analyzer 46 isolates the measurementscorresponding to regions of interest from the measurements correspondingto other regions of the test strip 50. The isolated measurements havehigher signal-to-noise ratios than aggregated measurements that includemeasurements from regions outside of the regions of interest.

The data analyzer 46 then computes at least one parameter from ones ofthe identified light intensity measurements (FIG. 4 , block 64).Exemplary parameters include peak intensity and aggregate intensityvalues. Since the measurements that are used to compute these parametershave higher signal-to-noise ratios, they characterize the region ofinterest with greater accuracy and, thereby, improve the results of thelateral flow assay.

B. An Exemplary Implementation of the Diagnostic Test System

FIG. 5A shows an exemplary implementation of the diagnostic test system40 that includes a light source 66, a two-dimensional array 68 of lightdetectors 70, and a lens 72. In FIG. 5A, the gross structural featuresof the test strip 50 are substantially the same as the correspondingfeatures the test strip 10, which is shown in FIG. 1 . In particular,the test strip 50 includes the sample receiving zone 12, the labelingzone 14, the detection zone 15, and the absorbent zone 20 on the commonsubstrate 22. In the illustrated implementation, a substantial portionof the detection zone 15 is exposed for optical inspection.

In operation, the light source 66 illuminates with light 76 the exposedportion of the detection zone 15, including the test region 16 and thecontrol region 18 of the test strip 50. The illuminating light 76 may bebroadband or narrowband and may be polarized or non-polarized. The lightdetector array 68 obtains separable localized light intensitymeasurements from the illuminated regions of the detection zone 15. Ingeneral, the light intensity measurements may be unfiltered or they maybe filtered in terms of at least one of wavelength and polarization. Thelight detector array 68 may be synchronized with the light source 66. Ingeneral, the light detector array 68 may measure light intensity whilethe detection zone 15 is being illuminated or after the light source 66has illuminated the detection zone 15. Light reflected or fluorescingfrom the detection zone 15 is focused by the lens 72 onto the individuallight detectors 70 of the light detector array 68. Each of the lightdetectors 70 receives light from a respective localized region of thedetection zone 15. That is, each light detector 70 is able to resolve orseparably image a respective localized region of the detection zone 15.In this implementation, the localized regions are characterized by asurface dimension that is at most as large as the narrowest dimension ofthe test and control regions 16, 18 (i.e., the dimensions of regions 16,18 that are along the lateral flow direction). In the illustratedimplementations, the localized regions are characterized by squaredimensions that are approximately equal to one-third of the size of thetest and control regions 16, 18 along the lateral flow direction. Thelight detectors 70 produce signals representative of the amount of lightreceived from the respective localized regions. These signals may bestored in a memory or they may be transmitted to the data analyzer 46for processing.

As shown in FIGS. 5A and 5B, the reflected or fluorescing light from thetest region 16 is received by only a subset 80 of the light detectors 70in the array 68. Similarly, the reflected or fluorescing light from thecontrol region 18 is received by only a subset 82 of the light detectors70 in the array 68. Thus, the signals from the light detectors in thesubsets 80, 82 provide relatively low noise light intensity measurementsof the light reflected or fluorescing from the test region 16 and thecontrol region 18, respectively.

The data analyzer 46 is operable to process the signals that aregenerated by the individual light detectors 70 to identify the ones ofthe light intensity measurements that are obtained from the regions ofinterest (e.g., the test region 16 and the control region 18). Referringto FIG. 6 , in one illustrative example, the light detector array 68produces a set of light intensity signals that are represented by athree-dimensional surface 84. In this example, the surface 84 includeshigher intensity measurements 86, 88 from locations of the detectionzone 15 corresponding to the subsets 80, 82 of the light detectors 70 inthe light detector array 68. With respect to this example, the dataanalyzer 46 may identify the light intensity measurements that areobtained from the test region 16 and the control region 18 bythresholding the surface 84 at an intensity threshold level 90. In someimplementations, the threshold that is used in the thresholding processis constant across the strip or region of interest. For example, in someimplementations, the threshold may be slanted or have local variationsto account for variations in illumination or diffusion of the analytes.The ones of the light intensity measurements that are above thethreshold level 90 are identified as having come from the test region 16and the control region 18. Additional information, such as the relativepositions of the light detector array 68 from which the identified onesof the light intensity measurements were obtained, may be used by thedata analyzer 46 to correlate the identified light intensitymeasurements with the test region 16 and the control region 18.

III. POSITION MARKERS ON A TEST STRIP AND READING SAME

A. Overview

In some embodiments, the test strip 50 includes one or more referencefeatures that serve as position markers, which are aligned with respectto regions of interest in the test strip. These embodiments enable thelevels of noise (e.g., noise caused by reflection of light or intrinsicfluorescence from materials in the test strip) in lateral flow assaymeasurements to be reduced by restricting the measurements to theregions of interest on the test strip based on measurements obtainedfrom the reference features. In this way, these embodiments increase thesignal-to-noise levels of these measurements and, thereby, increasemeasurement sensitivity and reduce the incidence of erroneous resultsfor low concentrations of analytes.

FIG. 7 shows an embodiment of a method by which the diagnostic testsystem 40 (FIG. 3 ) reads a test strip having at least one positionmarker.

In accordance with this method, the reader 44 obtains light intensitymeasurements from the test strip (FIG. 7 , block 92). In theimplementation shown in FIG. 5A, the light source 66 illuminates theexposed portion of the detection zone 15, including the test region 16and the control region 18 of the test strip 50, with light 76. Theilluminating light 76 may be broadband or narrowband and may bepolarized or non-polarized. The light detector array 68 then obtainsseparable localized light intensity measurements from the illuminatedregions of the detection zone 15.

The data analyzer 46 identifies ones of the light intensity measurementsthat are obtained from a region of interest (e.g., the test region 16 orthe control region 18) based on at least one measurement that isobtained from the at least one position marker (FIG. 7 , block 94). Inthis process, the data analyzer 46 may identify the ones of the lightintensity measurements that are obtained from a region of interest basedon predetermined information about the spatial relationship between theregion of interest and the at least one position marker.

In general, each of the position markers may be implemented by any typeof feature that has a different optical, electrical, or mechanicalcharacteristic than the adjacent regions of the test strip surface.

B. Optical Position Markers

In some implementations, an optical position marker may have adetectable optical response that is different from the optical responseof adjacent surface regions. For example, an optical position marker mayhave a greater reflection or emission than adjacent surface regions withrespect to light within a specified wavelength range (e.g., the visiblewavelength range: 390 nm to 770 nm). In other implementations, anoptical position marker may have a lower reflection or emission thanadjacent surface regions with respect to light within the specifiedwavelength range. In some implementations, the optical position markeris capable of fluorescent emission within a first wavelength range,whereas the adjacent surface regions are capable of fluorescent emissionwithin a second wavelength range different from the first wavelengthrange or with an intensity that is significantly lower than theintensity of fluorescent emission by the optical position marker withinthe first wavelength range.

FIG. 8 shows an embodiment of a method by which the data analyzer 46identifies ones of the light intensity measurements that are obtainedfrom a region of interest in the detection zone 15 based on intensitymeasurements that are obtained from at least one optical position markerthat is aligned with respect to the region of interest along the lateralflow direction 51.

In accordance with this embodiment, the data analyzer 46 identifies onesof the light intensity measurements that are obtained from the at leastone optical position marker (FIG. 8 , block 96).

The data analyzer 46 may identify the light intensity measurements thatare obtained from the at least one optical position marker in any of awide variety of different ways that depend on the implementation of theoptical position marker and the other regions in the detection zone. Asexplained above, each of the optical position markers may be implementedby any type of feature on a surface of the test strip 50 that has adifferent optical characteristic than the adjacent regions of the teststrip surface. In some implementations, the optical position markers arecomposed of quantum dots that exhibit fluorescent emission with narrowwavelength ranges or other optically recognizable media. The opticalposition markers may be formed on the exposed surface of the test strip50 in any of a wide variety of different ways, including silk screeningand other printing or deposition methods. The data analyzer 46 mayidentify the ones of the light intensity measurements that correspond tothe optical position marker by identifying the light intensitymeasurements that have one or more predetermined attributes, such asexhibiting a characteristic pattern of light intensity variations acrossthe test strip along the lateral flow direction or having the highestrelative intensities within a specified wavelength range.

After identifying the light intensity measurements that are obtainedfrom the at least one optical position marker (FIG. 8 , block 96), thedata analyzer 46 identifies ones of the light intensity measurementsthat are obtained from the region of interest based on a rulecorrelating imaged positions of the at least one optical position markerand the region of interest (FIG. 8 , block 98). In some implementations,the at least one optical position marker encodes at least one positionalong the lateral flow direction. In these implementations, the dataanalyzer 46 infers the imaged position of the region of interest basedon the positions that are encoded by the optical position markers. Forexample, the encoded position may correspond to one or both of thebeginning and ending locations of the region of interest along thelateral flow direction. In these implementations, the data analyzer 46may infer that the region of interest is located after, before orbetween the locations demarcated by the at least one optical positionmarker.

FIG. 9 shows an implementation of the test strip 50 that includes anexemplary set of optical position markers 100 that are spaced regularlyalong the edge of the test strip 50. The optical position markers 100include features that have a different reflection or emissioncharacteristic than the surface of the test strip 50. As a result, themeasurements that are obtained near the edge of the test strip 50 varyin intensity in accordance with the pattern of the optical positionmarkers 100. In this way, the optical position markers 100 encodepositions along the test strip 50 in the lateral flow direction 51. Withrespect to the implementation shown in FIG. 9 , the data analyzer 46 maydetermine the encoded positions along the lateral flow direction byincrementing a position counter with each intensity variation cycle(e.g., peak-to-valley) in the light intensity measurements obtained fromthe edge of the detection zone 15.

In these implementations, the data analyzer 46 correlates the lightintensity measurements with the positions along the test strip 50 in thelateral flow direction 51. The location correlation information may bestored in a lookup table that is indexed by the position counter value.Based on this information and on the predetermined informationcorrelating the locations of the regions of interest with the lightintensity contrast pattern produced by the optical position markers 100,the data analyzer 46 can identify the ones of the light intensitymeasurements corresponding to the regions of interest.

In other implementations, the optical position markers 100 may encodeposition information in variations in the lengths of the opticalposition markers along the lateral flow direction 51. Alternatively, theoptical position markers 100 may encode position information invariations in the spacing between adjacent ones of the optical positionmarkers 100 along the lateral flow direction 51.

FIG. 10 shows an implementation of the test strip 50 that includes anexemplary set of optical position markers 102, 104, 106, 108 that arepositioned adjacent to the test region 16 and the control region 18. Inparticular, the optical position marker 102 is positioned adjacent to anupstream edge 110 of the test region 16 and the optical position marker104 is positioned adjacent to a downstream edge 112 of the test region16. Similarly, the optical position marker 106 is positioned adjacent toan upstream edge 114 of the control region 18 and the optical positionmarker 108 is positioned adjacent to a downstream edge 116 of thecontrol region 18. In the illustrated embodiment, the optical positionmarkers 102-108 are beside one edge of the detection zone 15.

In the illustrated embodiment, the optical position markers 102-108 havesquare shapes. In general, however, the optical position markers 102-108may have any type of shape, including a polygonal (e.g., rectangular)shape and a curved (e.g., elliptical or circular) shape.

In some implementations, the data analyzer 46 is operable to identifythe light intensity measurements that are obtained from the opticalposition markers 102-108 based on the sizes, shapes, and/or locations ofthe optical position markers 102-108. For example, the data analyzer 46may identify the light intensity measurements by locating square regionsin an image of the detection zone 15 that is captured by the lightdetector array 68. In other implementations, the data analyzer 46 mayidentify the light intensity measurements that are obtained from theoptical position markers 102-108 based on one or more attributes (e.g.,relative intensity, wavelength, or decay profile) of the light reflectedor fluorescing from the optical position markers 102-108.

The data analyzer 46 readily may determine the bounds of the regions ofinterest 16, 18 based on the edges of the optical position markers102-108 in an image that is captured by the light detector array 68. Forexample, with respect to the implementation illustrated in FIG. 10 , thedata analyzer 46 identifies the test region 16 as the transverse regionbetween the optical position markers 102, 104 and identifies the controlregion 18 as the transverse region between the optical position markers106, 108. If only the upstream optical position markers 102, 106 werepresent, the data analyzer 46 would be configured to identify theregions of interest 16, 18 as corresponding to the transverse regionsimmediately following the optical position markers 102, 106 with respectto the lateral flow direction. Similarly, if only the downstream opticalposition markers 104, 108 were present, the data analyzer 46 would beconfigured to identify the regions of interest 16, 18 as correspondingto the transverse regions immediately preceding the optical positionmarkers 104, 108 with respect to the lateral flow direction 51.

In the implementation of the test strip 50 that is illustrated in FIG.10 , the optical position markers 102-108 are located beside an edge ofthe detection zone 15. FIG. 11 shows another implementation of the teststrip 50 in which the optical position markers 118, 120, 122, 124 arelocated centrally over the flow path in the detection zone 15. Theoptical position markers 118-124 are elongate in the transversedirection perpendicular to the lateral flow direction 51. In otherrespects, the optical position markers 118-124 may be implemented in thesame way as the optical position markers 102-108.

FIG. 12A shows an embodiment of the test strip 50 in which respectiveoptical position markers are formed by the spatial arrangement of theimmobilized test reagent in the test region 16 and the spatialarrangement of the immobilized test reagent in the control region 18. Inparticular, the test region 16 includes three discrete, spaced-apartcode areas 126, 128, 130 in which the test reagent is immobilized.Similarly, the control region 18 includes three discrete, spaced-apartcode areas 132, 134, 136 in which the control reagent is immobilized.Each of the code areas 126-136 has a rectangular shape with the samelength (L) but varying width. In this exemplary embodiment, each of thecode areas has a width that is an integer multiple of a unit width (w).For example, the widths of the code areas 126, 130, 134, and 136 areequal to the unit width w, whereas the widths of the code areas 128, 132are equal to 3w. The position information is encoded in the varyingwidths of the code areas of the test region 16 and the control region18.

FIG. 12B shows a graph of aggregate light intensity plotted as afunction of position along the lateral flow direction 51 of test stripshown in FIG. 12A. With respect to this example, the light detectorarray 68 of the diagnostic test system 20 shown in FIG. 5A captures afirst signature light code pattern 138 from the code areas 126-130 ofthe test region 16 and a second signature light pattern 140 from thecode areas 132-136 of the control region 18. The data analyzer 46determines the widths of the code areas 126-136 from the signature lightpatterns 138, 140 and translates the widths into code values thatuniquely identify the test region 16 and the control region 18. Forexample, from the signature light pattern 138, the data analyzer 46determines that the widths of the code areas 126-130 are w, 3w, and wand translates these widths into the code value 131. From the signaturelight pattern 140, the data analyzer 46 determines that the widths ofthe code areas 132-136 are 3w, w, and w and translates these widths intothe code value 311. In some embodiments, the code values identifying thetest region 16 and the control region 18 are stored in a lookup table,which the data analyzer 46 may query to determine the locations of thetest region 16 and the control region 18.

In general, the immobilized test reagent and the immobilized controlreagent may be arranged in any one-dimensional or two-dimensionalpattern that uniquely identifies the test region 16 and the controlregion 18, respectively. In some implementations, the immobilizedreagents of the test and control regions 16, 18 are spatially arrangedto form one- or two-dimensional bar code symbols. In some embodiments,the bar codes symbols are patterns of parallel bars and spaces ofvarious widths that represent data elements or characters. Typically,the bars represent strings of binary ones and the spaces representstrings of binary zeros. A one-dimensional bar code symbol (e.g., a UPCbar code symbol) typically contains a series of bars and spaces thatvary only in a single dimension. In two-dimensional bar codes (e.g., aPDF417 bar code symbol, a Code 1 bar code symbol, and a Maxicode barcode symbol), the bar code patterns vary in two dimensions. With respectthese embodiments, the diagnostic test system 40 implements the bar codedecoding process corresponding to the bar coding method that is used tocreate the code areas of the test and control regions 16, 18.

In addition to identifying the locations of the test and control regions16, 18 in the detection zone 15, the code areas may encode additionalinformation relating to the test strip in general (e.g., date ant placeof manufacture), the test and control regions in particular (e.g., thetype of target analyte that may be assayed), or to methods of readingthe test strip (e.g., predetermined calibration values or scaling valuesfor adjusting the measurement results or interpreting the measurementresults).

C. Electrical Position Markers

In some embodiments, the test strip 50 includes electrical positionmarkers that are aligned with respective regions of interest on the teststrip. With respect to these embodiments, the data analyzer 46identifies the ones of the light intensity measurements that areobtained from the respective regions of interest based on predeterminedinformation about the spatial relationship between the regions ofinterest and the corresponding electrical position markers.

FIG. 13A shows an implementation of the test strip 50 that includes anexemplary set of electrical position markers 140, 142, 144, 146 that arespaced to along the edge of the test strip 50. The electrical positionmarkers 140-146 include features that have a different electricalcharacteristic than the adjacent areas on the surface of the test strip50. As a result, the measurements that are obtained near the edge of thetest strip 50 vary in electrical response in accordance with the patternof the electrical position markers 140-146. With respect to theseembodiments, the diagnostic test system 40 (FIG. 3 ) includes adetection system that is capable of detecting the electrical positionmarkers. In general, any type of electrical conductor detection methodmay be used to detect the electrical position markers, includingcurrent, voltage, resistance, and capacitance based measurement methods.

FIG. 13B shows an embodiment of a detection system 148 on a portion ofan embodiment of the test strip shown in FIG. 13A. The detection system148 includes a detector 150, a first electrical contact 152, and asecond electrical contact 154. The first and second electrical contacts152, 154 are electrically connected to the detector 150 and areseparated from one another by an air gap 156, which forms an opencircuit. The detector 150 may include any type of circuit (e.g., anohmmeter, a voltmeter, and an ammeter) that is capable of detecting whenan electrical connection is formed across the air gap 156. In theseembodiments, the top surface of the test strip is formed of a materialwith a high electrical resistance except at the locations of theelectrical position markers 140-146.

In operation, at least one of the detection system 148 and the teststrip 50 is moved relative to the other in a direction parallel to thelateral flow direction 51. The first and second electrical contacts 152,154 slide over the top surface of the test strip. In someimplementations, the first and second electrical contacts 152, 154 areurged (e.g., by springs) against the top surface of the test strip. Inthe position shown in FIG. 13B, the first and second electrical contacts152, 154 are connected only by the material of the top surface of thetest strip. In this position, the detector 150 is configured todetermine that there is an open circuit between the first and secondelectrical contacts 152, 154. In the position shown in FIG. 13C, on theother hand, the first and second electrical contacts 152, 154 areconnected by the electrical position marker 146. In this position, thedetector 150 is configured to determine that there is a closed circuitbetween the first and second electrical contacts 152, 154.

The detector 150 may determine whether there is an open circuit or aclosed circuit between the first and second electrical contacts 152, 154by comparing an electrical measurement (e.g., current, voltage, orresistance) between the first and second electrical contacts 152, 164 toa threshold value. For example, the detector may determine that there isan open circuit between the first and second electrical contacts 152,154 when the measured electrical resistance value is greater than orequal to a threshold resistance value and that there is a closed circuitbetween the first and second electrical contacts 152, 154 when themeasured electrical resistance value is below the threshold value.

In the embodiment shown in FIG. 13A, the electrical position markers140-146 are aligned with the upstream and downstream edges of the testregion 16 and the control region 18 along the lateral flow direction 51.In this way, the data analyzer 46 readily may determine that the testand control regions 16, 18 are located between the detected positions ofthe electrical position markers 140, 142 and 144, 146, respectively.

In other embodiments, the electrical position markers may encodeposition information in different ways. For example, in someembodiments, the electrical position markers may be positioned atregularly spaced locations along the edge of the test strip 50. As aresult, the electrical measurements that are obtained near the edge ofthe test strip 50 vary in value in accordance with the pattern of theelectrical position markers. In this way, the electrical positionmarkers encode positions along the test strip 50 in the lateral flowdirection 51. With respect to these embodiments, the data analyzer 46may determine the encoded positions along the lateral flow direction 51by incrementing a position counter with each measurement variation cycle(e.g., peak-to-valley) in the electrical measurements obtained from theedge of the detection zone 15.

D. Mechanical Position Markers

In some embodiments, the test strip 50 includes mechanical positionmarkers that are aligned with respective regions of interest on the teststrip. With respect to these embodiments, the data analyzer 46identifies the ones of the light intensity measurements that areobtained from the respective regions of interest based on predeterminedinformation about the spatial relationship between the regions ofinterest and the corresponding mechanical position markers.

FIG. 14A shows an implementation of the test strip 50 that includes anexemplary set of mechanical position markers 160, 162, 164, 166 that arespaced along the edge of the test strip 50. The mechanical positionmarkers 160-166 include features that have a different surface profilethan the adjacent areas on the surface of the test strip 50. As aresult, the measurements that are obtained near the edge of the teststrip 50 vary in mechanical response in accordance with the pattern ofthe mechanical position markers 160-166. With respect to theseembodiments, the diagnostic test system 40 (FIG. 3 ) includes adetection system that is capable of detecting the mechanical positionmarkers. In general, any type of detection method that is responsive tovariations in surface profile may be used to detect the mechanicalposition markers, including spring contact based methods and mechanicaltransducer based methods.

FIG. 14B shows an embodiment of a detection system 168 on a portion ofan embodiment of the test strip shown in FIG. 14A. The detection system168 includes a detector 170, a first electrical conductor 172, and asecond electrical conductor 174. The first and second electricalconductors 172, 174 are electrically connected to the detector 170 andare separated from one another by the substrate of the test strip 50.The first electrical conductor 172 is attached to a spring-loaded piston176 and the second electrical conductor 174 is an electricallyconducting support member for the test strip 50. In other embodiments,the first electrical conductor 172 may be implemented by an electricallyconducting brush electrode. The detector 170 may include any type ofcircuit (e.g., an ohmmeter, a voltmeter, and an ammeter) that is capableof detecting when the first and second electrical conductors areelectrically connected together. In these embodiments, the test strip isformed of a material with a high electrical resistance and themechanical position markers 160-166 correspond to respective holes thatextend through the substrate 22 of the test strip 50.

In operation, at least one of the detection system 168 and the teststrip 50 is moved relative to the other in a direction parallel to thelateral flow direction 51. The first electrical conductor 172 slidesover the top surface of the test strip 50, which is supported by thesecond electrical conductor 174. The first electrical conductor 172 isurged by a spring 177 against the top surface of the test strip 50. Inthe position shown in FIG. 14B, the first and second electricalconductors 172, 174 are connected only by the high resistance materialof the test strip 50. In this position, the detector 170 is configuredto determine that there is an open circuit between the first and secondelectrical conductors 172, 174. In the position shown in FIG. 14C, onthe other hand, the first electrical conductor 172 extends through themechanical position marker 166 and directly contacts the secondelectrical conductor 174. In this position, the detector 170 isconfigured to determine that there is a closed circuit between the firstand second electrical conductors 172, 174.

The detector 170 may determine whether there is an open circuit or aclosed circuit between the first and second electrical conductors 172,174 by comparing an electrical measurement (e.g., current, voltage, orresistance) between the first and second electrical conductors 172, 174to a threshold value. For example, the detector may determine that thereis an open circuit between the first and second electrical conductors172, 174 when the measured electrical resistance value is greater thanor equal to a threshold resistance value and that there is a closedcircuit between the first and second electrical conductors 172, 174 whenthe measured electrical resistance value is below the threshold value.

In the embodiment shown in FIG. 14A, the mechanical position markers160-166 are aligned with the upstream and downstream edges of the testregion 16 and the control region 18 along the lateral flow direction 51.In this way, the data analyzer 46 readily may determine that the testand control regions 16, 18 are located between the detected positions ofthe mechanical position markers 160, 162 and 164, 166, respectively.

In other embodiments, the mechanical position markers may encodeposition information in different ways. For example, in someembodiments, the mechanical position markers may be positioned atregularly spaced locations along the edge of the test strip 50. As aresult, the electrical measurements that are obtained near the edge ofthe test strip 50 vary in value in accordance with the pattern of themechanical position markers. In this way, the mechanical positionmarkers encode positions along the test strip 50 in the lateral flowdirection 51. With respect to these embodiments, the data analyzer 46may determine the encoded positions along the lateral flow direction 51by incrementing a position counter with each measurement variation cycle(e.g., peak-to-valley) in the electrical measurements obtained from theedge of the detection zone 15.

In other embodiments, the first and second electrical conductors 172,174 of the detection system 168 are replaced by a mechanical transducer(e.g., a stylus connected to a piezoelectric element) is dragged acrossthe top surface of the test strip. In particular, the mechanicaltransducer generates signals corresponding to its movement in adirection normal to the surface of the test strip. The vertical motionof the stylus compresses the piezoelectric element, which generates avoltage response that varies linearly with the movement of the stylus.These signals indicate the surface profile variations across the surfaceof the test strip 50. With respect to these embodiments, the mechanicalposition markers may be implemented by holes, notches, dimples, or bumpson the top surface of the test strip 50.

III. CALIBRATION REGIONS ON A TEST STRIP AND READING SAME

A. Overview

In some embodiments, one or more of the reference features on the teststrip 50 are calibration regions that provide a reference opticalresponse that may be used by embodiments of the diagnostic test system10 to calibrate one or more components of a diagnostic test system andthe assay measurements obtained by such a system and, thereby, increasethe accuracy of the lateral flow assay results.

In general, the calibration regions may be laid out in the detectionzone 15 of the test strip 50 in any of a wide variety of ways. In someimplementations, the calibration regions are positioned near the regionsof interest in order to reduce the effects of temperature ormanufacturing variations across the test strip 50. In some of theseimplementations, the calibration regions are laid out adjacent to one ormore of the regions of interest in the detection zone 15 of the teststrip 50. For example, in one exemplary embodiment, the calibrationregions may be laid out in the same way as the optical position markers102-108 in the implementation shown in FIG. 10 or the same way as theoptical position markers 118-124 in the implementation shown in FIG. 11.

In implementations of the test strip 50 that include a test region andone or more additional capture regions (e.g., another test region or acontrol region), one or more the additional capture regions may serve asa calibration region for calibrating one or more components of adiagnostic test system and/or calibrating the assay measurementsobtained from the test region.

B. Measurement Calibration Regions

In some embodiments, the calibration regions are calibrated to provide areference optical response that may be used by the data analyzer 46 tocalibrate the light intensity measurements that are obtained from theregions of interest. FIG. 15 shows an embodiment of a method by whichthe diagnostic test system 10 calibrates the light intensitymeasurements that are obtained from a region of interest.

In accordance with this method, the data analyzer 46 determines ameasurement calibration value from at least one light intensitymeasurement that is obtained from a calibration region (FIG. 15 , block180). The measurement calibration value may correspond to a statisticalmeasure (e.g., a peak intensity value or average intensity value) thatis computed from the light intensity measurements that are obtained fromthe calibration region.

The data analyzer 46 then determines an amount of a target substance(e.g., the target analyte captured by the test region 16 or the labelcaptured by the control region 18) based on the light intensitymeasurements that are obtained from the region of interest and thedetermined measurement calibration value (FIG. 15 , block 182).

In some implementations, at least one calibration region contains acalibrated amount of the test label 32 such that the optical response(e.g., reflected light intensity or fluorescent emission intensity) ofthe calibration region corresponds to a known quantity (e.g., number ordensity) of the test label 32. The data analyzer 46 may then scale theoptical responses from the test region 16 and the control region 18based on the optical response of the calibration region to obtainmeasures of the quantity of the analyte captured by the test region 16or the quantity of the label captured by the control region 18.

In some implementations, the test strip 50 includes multiple calibrationregions having different respective calibrated amounts of the samelabel. With respect to these implementations, the data analyzer 46generates from the optical responses of the calibration regions acalibration curve mapping light intensities to measures of thequantities of the label. The data analyzer 46 may then map the lightintensity measurements that are obtained from the regions of interest toa measure of the amount of a captured substance of interest (e.g., thetarget analyte captured in the test region 16 or the label captured bythe control region 18).

In some implementations, the labeling zone 14 of the test strip 50contains different colored labels that specifically bind to differentrespective analytes that may be present in the fluid sample. In theseimplementations, the test strip 50 may include one or more calibrationregions that contain calibrated amounts of the different labels. Inthese implementations, the test strip 50 may include a separatecalibration region for each label. Alternatively, the test strip 50 maycontain one or more calibration regions each of which containscalibrated amounts of multiple different labels. The calibrated amountsof the different labels may be intermixed and distributed across thesame calibration region or they may be located within differentrespective sub-areas of the same calibration region.

C. Detection Calibration Regions

In some embodiments, the calibration regions provide a reference opticalresponse that may be used by the data analyzer 46 to calibrate one ormore operational parameters of the detection system of the reader 44.

In this regard, the data analyzer 46 may optimize the wavelengthcharacteristics of the components of the detection system thatdistinguish among the different light colors that are emitted bydifferent labels on the test strip 50. For example, the data analyzer 46may generate a signal that adjusts the wavelength passband of a tunableoptical filter of the detection system to maximize the detectedintensity of light received from a calibration region.

The data analyzer 46 also may adjust (e.g., normalize) the response ofthe detection system for the characteristic wavelengths of lightreceived from the calibration regions. For example, the data analyzer 46may generate a signal that adjusts the response of the detection systemso that it produces a predetermined output value (e.g., a predeterminedcurrent value or a predetermined voltage value) in response to lightobtained from the calibration region. The data analyzer 46 may generatea respective response adjustment signal for each wavelength range ofinterest (e.g., for the characteristic wavelength of each label carriedby the test strip 50).

D. Illumination Calibration Regions

In some embodiments, the calibration regions provide a reference opticalresponse that may be used by the data analyzer 46 to calibrate one ormore operational parameters of the illumination system of the reader 44.

FIG. 16 shows an embodiment of the test strip 50 that includes first andsecond illumination source calibration regions 184, 186. Each of thefirst and second illumination source calibration regions 184, 186 has areflection or emission that is greater than the adjacent surface regionsof the test strip with respect to light within a target wavelengthrange. In some implementations, the first and second illumination sourcecalibration regions 184, 186 have reflectivities that are greater than90% with respect to light within the visible wavelength range (i.e., 390nm to 770 nm). In these implementations, the first and secondillumination source calibration regions 184, 186 are formed of thinfilms of a metal (e.g., aluminum or gold). In other implementations, thefirst and second illumination source calibration regions 184, 186include immobilized fluorescent particles (e.g., quantum dots) withsecondary fluorescent emissions that may be used to calibrate one ormore operational parameters of the illumination system of the reader 44.

FIG. 17 shows an embodiment of a method by which the data analyzer 46calibrates an illumination source of the reader 44.

In accordance with this method, the data analyzer 46 determines anillumination source output measure from at least one light intensitymeasurement obtained from the illumination source calibration region(FIG. 17 , block 190). In some implementations, the illumination sourcecalibration source corresponds to a statistical measure (e.g., a peakintensity value or average intensity value) that is computed from thelight intensity measurements that are obtained from the illuminationsource calibration region.

The data analyzer 46 generates a signal for calibrating the illuminationsource based on the illumination source output measure (FIG. 17 , block192). In some implementations, the data analyzer 46 compares theillumination source output measure to a reference value. The dataanalyzer 46 may generate a control signal that increases the lightintensity output of the illumination source when the illumination sourceoutput measure is below the reference value and decreases the lightintensity output of the illumination source when the illumination sourceoutput measure is above the reference value. In some implementations,the data analyzer 46 may iteratively determine the illumination sourceoutput measure and generate the illumination control signal until theillumination source output measure is within a specified range of thereference value.

In the embodiment shown in FIG. 16 , the first illumination sourcecalibration region 184 is located upstream of the test region 16 and thesecond illumination source calibration region 186 is located downstreamof the control region 18. In this way, the data analyzer 46 may detectvariations in the output of the illumination source across the detectionzone 15. In some implementations, the data analyzer 46 may scale thelight intensity measurements obtained for the regions of interest basedon the differences between the illumination source output measuresdetermined for the first and second illumination source calibrationregions 184, 186. The amount by which the light intensity measurementsare scaled may be determined empirically and stored in a lookup table orrepresented by a parametric curve or some other function of theillumination source output measures.

IV. CONCLUSION

The embodiments that are described above provide lateral flow assay teststrips that have one or more reference features. In some embodiments,the reference features are position markers that are aligned withrespect to regions of interest in the test strip and may be used byembodiments of the diagnostic test system to identify light intensitymeasurements obtained from regions of interest. In some embodiments, thereference features are calibration regions that provide a referenceoptical response that may be used by embodiments of the diagnostic testsystem to calibrate one or more components of a diagnostic test systemand the assay measurements obtained by such a system. In these ways, theembodiments described above improve the accuracy and precision withwhich analytes in a fluid sample may be assayed.

Other embodiments are within the scope of the claims.

For example, the embodiments are described above in connection with animplementation of the diagnostic test system 10 that includes atwo-dimensional array of light detectors 70. These embodiments also maybe integrated with different implementations of the diagnostic testsystem 10, including implementations in which the reader 44 includes aone-dimensional array of light detectors and a mechanism for impartingrelative motion between the optical inspection components of the readerand the test strip-.

Some implementations of the test strip 50 may include two or more of thedifferent types of reference features that are described above.

In the embodiments described above, the test regions, control regions,calibration regions, and position markers are shown as havingrectangular shapes in the plane of the detection zone 15. In general,however, these features may have any type of shape, including apolygonal (e.g., rectangular) shape and a curved (e.g., elliptical orcircular) shape.

What is claimed is:
 1. A diagnostic test system, comprising: a housing comprising a port constructed and arranged to receive a test strip, and configured to enclose a reader and a data analyzer; a test strip configured to be loaded into the port, the test strip comprising a flow path for a fluid sample, a sample receiving zone coupled to the flow path, a labeling zone comprising a label that specifically binds a target analyte, a detection zone coupled to the flow path and comprising a test region exposed for optical inspection and having an immobilized test reagent that specifically binds the target analyte, and at least one reference feature on a surface of the test strip; a reader comprising an illumination source configured to illuminate the test strip when the test strip is loaded in the port, the reader further comprising a detection system configured to detect the at least one reference feature and to obtain light intensity measurements from exposed regions of the test strip when the test strip is loaded in the port, the reader configured to illuminate a second test strip with an adjusted light intensity output of the illumination source when the second test strip is loaded in the port; and a data analyzer comprising software configured to (a) identify at least one light intensity measurement obtained from the test region based on at least one measurement obtained from the at least one reference feature, (b) determine an illumination source output measure from the at least one measurement obtained from the at least one reference feature, and (c) adjust a light intensity output of the illumination source when the determined illumination source output measure is different than a reference value, the at least one reference feature comprising a plurality of position markers aligned with respect to a region of interest in the test strip, the reader further configured to obtain from the plurality of position markers a series of light intensity measurements encoding positions along the flow path, the data analyzer comprising software configured to identify the at least one light intensity measurement obtained from the test region based on the positions of the series of light intensity measurements.
 2. The system of claim 1, wherein the at least one reference feature comprises a plurality of optical, electrical, or mechanical markers.
 3. The system of claim 1, wherein the data analyzer comprises software configured to identify the at least one light intensity measurement obtained from the test region by correlating detection of the at least one reference feature by the detection system with the obtaining of the light intensity measurements by the reader.
 4. The system of claim 1, wherein the at least one reference feature comprises a plurality of electrical conductors.
 5. The system of claim 1, wherein the at least one reference feature is adjacent to the test region.
 6. The system of claim 1, wherein the data analyzer comprises software configured to determine an amount of the analyte captured in the test region based on at least one light intensity measurement obtained from the at least one reference feature and at least one light intensity measurement obtained from the test region.
 7. The system of claim 1, wherein: the at least one reference feature comprises a first reference feature upstream of and adjacent to the test region, and a second reference feature downstream of and adjacent to the test region; and the data analyzer comprises software configured to determine a measurement calibration value from light intensity measurements obtained from the first and second reference features and to determine an amount of the analyte based on the light intensity measurements obtained from the test region and the determined measurement calibration value.
 8. The system of claim 1, wherein the plurality of position markers is in an illumination source calibration region.
 9. The system of claim 1, wherein the data analyzer comprises software configured to generate a control signal modifying at least one operational parameter of the reader based on at least one measurement obtained from the at least one reference feature, wherein the at least one operational parameter modified by the control signal comprises wavelength components of the reader operable to obtain light intensity measurements from exposed regions of the test strip, and wherein the data analyzer further comprises software configured to adjust the wavelength components of the reader in response to the generated control signal.
 10. The system of claim 1, wherein the data analyzer comprises software configured to generate a control signal modifying at least one operational parameter of the reader based on at least one measurement obtained from the at least one reference feature, wherein the at least one operational parameter modified by the control signal comprises a wavelength passband of a tunable optical filter, and wherein the data analyzer further comprises software configured to adjust the wavelength passband in response to the generated control signal.
 11. The system of claim 1, wherein the data analyzer comprises software configured to generate a control signal modifying at least one operational parameter of the reader based on at least one measurement obtained from the at least one reference feature, wherein the at least one operational parameter modified by the control signal comprises a response of the reader that has been normalized to produce a predetermined output value, and wherein the data analyzer further comprises software configured to adjust the predetermined output value in response to the generated control signal.
 12. The system of claim 1, further comprising the test strip loaded in the port of the reader, wherein the at least one reference feature comprises a film of metal or immobilized fluorescent particles.
 13. The system of claim 1, wherein the data analyzer comprises software configured to increase the light intensity output of the illumination source when the determined illumination source output measure is below the reference value, and to decrease the light intensity output of the illumination source when the determined illumination source output measure is above the reference value. 